Field tests were conducted on a late full sized sedan with the HVAC unit operating in both Recirculation and OSA modes to monitor build-up of the CO2 concentration inside the cabin and its influence on occupant’s fatigue and alertness. These tests were conducted during 2015 summer on interstate highways with test durations ranging from 4 to 7 hours. During the above tests, fatigue or tiredness of the occupants (including CO2 levels) was monitored and recorded at 30 min intervals.Based on this investigation it is determined that the measured cabin concentration levels reaches ASHRAE (Standard 62-1999) specified magnitudes (greater than 700 ppm over ambient levels) with three occupants in the vehicle. Further, the occupants did show fatigue when the HVAC unit was operated in recirculation mode in excess of 5 hours. Further details have been presented in the paper.